PROJECT SUMMARY Tuberculosis currently afflicts nearly one-third of the world's population, but progress in development of interventions is hamstrung by the slow growth rate of Mycobacterium tuberculosis, the causative agent, and the need to evaluate viability by colony forming units (cfu). The proposed study is designed to overcome this major roadblock through the development and validation of imaging technologies that can be applied to evaluation of novel prevention and treatment strategies for tuberculosis. Our previous studies have resulted in development of both fluorescent and bioluminescent imaging strategies for tuberculosis, but the most sensitive of these has proven to be reporter enzyme fluorescence (REF), which has a detection threshold of approximately 104 cfu during pulmonary infection. We have also shown that REF imaging allows therapeutic evaluation within 24-48 h post-treatment both in vitro and in animals. This work has set the foundation for the proposed studies designed to allow application of REF imaging to therapeutic efficacy determination, tracking Mtb infections in animals and analysis of vaccination strategies for tuberculosis. We have demonstrated the ability of imaging to quantify and track both pulmonary and subcutaneous infections, offering the unique opportunity to track both vaccination and challenge doses for tuberculosis, which has not been previously feasible, particularly in real-time using live animals. In the current study, we will build upon our earlier studies by testing the ability of REF imaging to evaluate efficacy of a diverse set of therapeutics, improvement of the current REF imaging threshold of detection and demonstrate the utility of imaging study of vaccine efficacy. Specifically, we propose to: 1) Improve REF imaging to facilitate tuberculosis research. Our working hypothesis is that the catalytic nature of REF imaging will allow this technology to achieve thresholds of 10 cfu per organ, sufficient for analysis of tuberculosis virulence and therapeutics. Our preliminary studies demonstrate that REF imaging is very sensitive, imaging can be used to measure therapeutic outcome and that there is potential to improve existing REF substrates. In this aim we will compare therapeutic efficacy evaluation for imaging with cfu, analyze and improve substrates for thresholds of detection and carefully evaluate our ability to accurately track Mtb infection in animal models. 2) Analyze vaccine efficacy against tuberculosis using imaging. Our working hypothesis is that the combination of micro-endoscopy and REF will allow us to evaluate tuberculosis vaccine efficacy more rapidly and in more detail than conventional cfu-based methods. Our preliminary studies demonstrate that we can utilize micro- endoscopic imaging to follow sub-cutaneous inoculation with reporter strains of the vaccine and REF imaging for pulmonary infection with Mtb. In this aim we will construct stable reporter vaccine strains and use them to follow both the vaccination and challenge dose throughout vaccination in mice and guinea pigs to better understand the live vaccine and challenge dose viability dynamics during vaccination.